Sequence analysis and prokaryotic expression system construction of PIA genes isolated from Neisseria gonorrhoeae
- VernacularTitle:淋病奈瑟菌临床菌株外膜蛋白PIA基因序列分析及原核表达系统的构建
- Author:
Aihua SUN
;
Chunhan SONG
;
Senlin WU
;
Yafei MAO
;
Haiou ZHOU
;
Jie YAN
- Publication Type:Journal Article
- Keywords:
Neisseria gonorrhoeae;
Genes, PIA;
Sequence analysis;
Prokaryotic expression
- From:
Chinese Journal of Pathophysiology
2000;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N. gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N. gonorrhoeae were amplified by using high fidelity PCR. The target amplification fragments were sequenced after T-A cloning. Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed. A prokaryotic expression system of PIA gene was constructed. Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA. rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962), the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%, respectively, which indicated that all the isolates were belonging to serovars IA6. Output of rPIA was as high as 50.1% of the total bacterial proteins. The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N. gonorrhoeae isolates and sequences of the encoding gene are relatively conserved. The constructed prokaryotic expression system is able to express rPIA with high efficiency, which may lay a foundation for further development of serological detection kit and vaccine of N. gonorrhoeae.
